The serine/threonine kinase Mirk/dyrk1B is expressed in about 90% of pancreatic ductal adenocarcinomas and in most pancreatic cancer cell lines where it mediates their clonogenic growth. Mirk may provide some survival advantage for pancreatic cancer cells as the Mirk gene was among 16 genes within the consistently amplified 660 kb subregion of the 19q13 amplicon in pancreatic cancers, while the nearby gene Akt2 was not. Mutant K-ras is the initiating lesion in most pancreatic cancers and leads to constitutive activation of Mirk kinase through the mutant K-ras/Rac1/MKK3 signaling pathway in pancreatic cancer cell lines. Thus Mirk is widely expressed in pancreatic cancers and mediated pancreatic cancer cell survival in clonogenic assays, while the Mirk gene was amplified in a subset of pancreatic cancers. Mirk was recently shown to have two primary functions in cancer cells. While cancer cells often exhibit higher levels of ROS than normal cells because of increased metabolism and oncogenic stimulation, Mirk lowered ROS levels, at least in part by increasing expression of antioxidant genes. The second function of Mirk in cancer cells was to work together with p27kip1 to block the exit of cells from G0. We adapted a two-parameter flow cytometry assay to measure DNA and RNA (predominately ribosomal) content independently, and found that all cultures of pancreatic cancer cells examined had at least 5% of cells in a G0 phase with a 2N DNA content and a low RNA content. The G0 fraction increased over 10-fold when cells were cultured under suboptimal conditions, which also elevated Mirk levels up to 10 fold. Mirk was a kinase most abundant and active in G0. Both the ROS suppressing function of Mirk and its capacity to limit cell cycling are unusual capacities to be combined in one kinase, particularly one which was shown not to be an essential gene in embryonic mouse knock-out studies. We hypothesize that cycling cancer cells may transiently enter G0 to repair damage incurred by oxidative stress from their elevated metabolism, and then re-enter cycle under favorable environmental conditions. The specific aims are (1) to test whether depletion of Mirk in cancer cells in G0 leaves cells with unrepaired DNA damage, sensitizing them to drugs which also damage DNA, to determine the types of ROS which accumulate in Mirk-depleted cells, and to extend the results of qRT-PCR assays which identified a small group of antioxidant genes as targets of upregulation by Mirk in SU86.86 cells to several additional pancreatic cancer cell lines. (2) Two human pancreatic cancer cell lines with a doxycycline-inducible shRNA to Mirk will be used as in vivo xenograft models in SCID/NOD mice to assess the effect of Mirk depletion alone, or together with treatment with either gemcitabine or with the natural compound PEITC (beta-phenylethyl isothiocyanate), which is known to raise ROS levels. PUBLIC HEALTH RELEVANCE: Pancreatic ductal adenocarcinoma is the fourth leading cause of cancer deaths in the U.S. A large fraction of these cancer cells in vivo are noncycling. Some of these cells may be reversibly blocked in a quiescent G0 state and can re-enter the cell cycle under favorable clues from the microenvironment, enabling tumor spread. Noncycling quiescent cancer cells are more resistant than cycling cells to most chemotherapeutic drugs and radiation. Factors which allow the prolonged survival of quiescent tumor cells in vivo are of clinical relevance, and include antioxidant proteins and other factors which repress the production of reactive oxygen species (ROS). The serine/threonine kinase Mirk is upregulated in quiescent pancreatic cancer cells and mediates their survival by increasing transcription of a cohort of antioxidant genes which detoxify superoxides and which prevent the generation of hydroxyl radicals. The proposed studies will document the effect of combining Mirk depletion with drugs that raise ROS levels, define a larger set of antioxidant genes upregulated by Mirk, measure DNA damage caused by Mirk depletion, and create murine models to define Mirk's actions in vivo.